Analytical apparatus

ABSTRACT

An analytical apparatus comprises a biosensor device (3) which forms the base of a sample chamber. A stirrer (8) extends into the sample chamber and moves within the chamber so as to homogenize a sample contained within the chamber in contact with the biosensor (3). Movement of the stirrer (8) is preferably reciprocal movement along an axis perpendicular to the surface of the biosensor (3).

This invention relates to analytical apparatus for the qualitative orquantitative determination of chemical or biochemical species or theirinteractions.

Many devices for the automatic determination of biochemical analytes insolution have been proposed in recent years. Typically, such devices(biosensors) include a sensitised coating layer which is located in theevanescent region of a resonant field. Detection of the analytetypically utilizes optical techniques such as, for example, surfaceplasmon resonance (SPR), and is based on changes in the thickness and/orrefractive index of the coating layer resulting from interaction of thatlayer with the analyte. This causes a change, eg in the angular positionof the resonance.

Other optical biosensors include a waveguide in which a beam of light ispropagated. The optical characteristics of the device are influenced bychanges occurring at the surface of the waveguide. One form of opticalbiosensor is based on frustrated total reflection. The principles offrustrated total reflection (FTR) are well known; the technique isdescribed, for example, by Bosacchi and Oehrle Applied Optics (1982),21, 2167-2173!. An FTR device for use in immunoassay is disclosed inEuropean Patent Application No 0205236A and comprises a cavity layerbounded on one side by the sample under investigation and on the otherside by a spacer layer which in turn is mounted on a substrate. Thesubstrate-spacer layer interface is irradiated with monochromaticradiation such that total reflection occurs, the associated evanescentfield penetrating through the spacer layer. If the thickness of thespacer layer is correct and the incident parallel wave vector matchesone of the resonant mode propagation constants, the total reflection isfrustrated and radiation is coupled into the cavity layer. The cavitylayer must be composed of material which has a higher refractive indexthan the spacer layer and which is transparent at the wavelength of theincident radiation.

In all such devices, problems can occur due to thermal effects. Foraccurate results it is vital to ensure that the sample reaches goodequilibration with the surroundings and that comparative measurementsare carried out at constant temperatures. In addition, inhomogeneitiesor transport phenomena occurring within the sample may lead todifficulties.

There has now been devised an analytical apparatus which overcomes orsubstantially mitigates the above-mentioned disadvantages.

According to the invention, there is provided an analytical apparatuscomprising a biosensor device, a sample chamber adjacent the biosensordevice, a stirrer extending into the sample chamber, and means forcausing the stirrer to move within the sample chamber.

The apparatus according to the invention is advantageous primarily inthat the motor-driven stirrer provides virtually instantaneoushomogeneity and uniformity of the samples, in terms of both compositionand temperature. This enables a larger area of sensitised coating to beused, which in turn leads to higher sensitivity. The apparatus offerssignificant advantages compared to known systems in which the samplechamber is a flow cell into which the sample is pumped, since in suchsystems the reaction kinetics are strongly dependent upon, and oftenadversely influenced by, the flow hydrodynamics.

The apparatus according to the invention is useful in the qualitative orquantitative determination of an analyte species in a sample or theirinteractions. The apparatus may be used not only for the determinationof the presence and/or concentration of a particular molecular species,but also to monitor any process in which the molecular species interactswith the surface of the biosensor or with other molecular species at orin the vicinity of the surface. For example, the parameter underinvestigation may be the binding affinity of a molecular species withthe biosensor surface.

Generally, the stirrer will comprise an elongate stirrer shaft. Theshaft may terminate at a point within the sample chamber which, in use,lies within the sample fluid. Alternatively, the shaft may be connectedto a further component which intrudes into the sample chamber.

The means for causing the stirrer to move within the sample chamber ispreferably an electric or electromagnetic motor. For some applications,eg applications in which particularly high frequencies are required,piezoelectric devices may be suitable.

The movement imparted to the stirrer may be rotary movement. In such acase, the portion of the stirrer which is, in use, immersed in thesample is preferably provided with a suitable paddle element. The paddleelement may take any form suitable for causing effective homogenisationof the sample. Most preferably, the paddle element rotates in a planeparallel to the sensitised surface of the biosensor device. Theclearance between the paddle element and the sensitised surface ispreferably less than 1 mm, more preferably less than 0.5 mm, eg about0.2 mm.

Preferably, however, the stirrer is not a rotary stirrer but a stirrerwhich vibrates. Most preferably, the motion of the stirrer isreciprocal, eg along an axis essentially perpendicular to the sensitisedsurface of the biosensor.

Again, the portion of the stirrer which, in use, is immersed in thesample is preferably provided with a suitable element to facilitatemixing of the sample. In one preferred embodiment, such an element takesthe form of a hollow truncated cone, the top and bottom faces of whichare open.

The means for imparting reciprocating motion to the stirrer element arepreferably electromagnetic. Most preferably, the upper end of thestirrer shaft is rigidly connected to a former on which is wound a wirecoil. The former surrounds a permanent magnet such that when analternating current is applied to the coil the former, and hence theshaft, oscillate at the frequency of the applied current.

In order to hold the former in position, and to limit the extent of thereciprocating movement, the shaft/former assembly is preferably securedto a flexible membrane which is held in a fixed position relative to themagnet.

The frequency of reciprocation of the stirrer element is typically ofthe order of a few tens to a few hundred Hertz. The frequency may, forexample, be up to about 250 Hz, typically 100-150 Hz.

The stirrer element preferably reciprocates over a distance of less than±1 mm, more preferably less than ±0.5 mm. The rest position of thestirrer element is preferably such that the separation of the stirrerelement from the sensitised surface of the biosensor device, at thepoint of closest approach, is less than 0.5 mm. For example, the restposition may be 0.5 mm above the surface and the extent of thereciprocal motion may be ±0.3 mm so that the stirrer element oscillatesbetween extreme positions 0.2 mm and 0.8 mm above the surface.

It is particularly preferred that the stirrer element should be capableof being switched off, if desired, at any stage of the measurementprocess ie the movement of the stirrer element should be capable ofbeing stopped. It is also preferred that the rest position and/oramplitude of modulation and/or frequency of movement of the stirrerelement should be adjustable to suit the particular sample underinvestigation.

It is preferred that the sample chamber forms part of a disposablecuvette.

The cuvette, or at least that part of the cuvette adjacent to the samplechamber, is preferably of a material with high thermal conductivity.

By "high thermal conductivity" is meant sufficient conductivity toprovide rapid transfer of heat from a supporting body against which thecuvette is placed. Suitable materials include metals.

Preferably, the cuvette body is of aluminium, more preferably aluminiumwith an inert coating such as electroless nickel, a fluorocarbon, or asilicon lacquer. In such a case the coating may be of the order of 25 μmin thickness.

The body is preferably provided with wings or flanges which provideintimate thermal contact with a temperature-controlled block on theanalytical apparatus, the relatively large surface area of the wings orflanges providing for rapid thermal equilibration between the block andthe body. For the same purpose the analytical apparatus preferablycomprises clamping means for holding the cuvette in firm contact withthe block.

Thus, according to another aspect of the invention there is provided asample cuvette comprising a body of material with high thermalconductivity, the body defining a sample chamber with a baseincorporating a sensitive surface of a biosensor device, and the bodybeing provided with integrally formed wings or flanges by which thecuvette may be held in intimate thermal contact with a support.

The cuvette preferably includes one or more reserve fluid wells formaintaining a supply of sample or analytical reagents at the measuringtemperature.

The volume of the sample chamber is preferably small, eg less than 500μl, and most preferably 100-300 μl.

The arrangement is preferably such that the thermal capacity of thecuvette is considerably greater than that of the fluid in the samplechamber.

The cuvette may contain more than one sample chamber. Such chambers maybe completely separate compartments, or may be formed in a single wellwith suitable partitions.

An advantage of the use of such a cuvette is that it may be readilyremoved from the analytical apparatus and replaced. A range of cuvettescontaining biosensors adapted for different types of measurement maytherefore be used, switching from one type of measurement to anotherbeing readily accomplished.

The biosensor device is preferably an FTR sensor. Such a sensor willgenerally include an optical structure comprising

a) a cavity layer of transparent dielectric material of refractive indexn₃,

b) a dielectric substrate of refractive index n₁, and

c) interposed between the cavity layer and the substrate, a dielectricspacer layer of refractive index n₂.

In use, the interface between the substrate and the spacer layer isirradiated with light such that internal reflection occurs. Resonantpropagation of a guided mode in the cavity layer will occur, for a givenwavelength, at a particular angle of incidence of the excitingradiation.

The angular position of the resonant effect depends on variousparameters of the sensor device, such as the refractive indices andthicknesses of the various layers. In general, it is a prerequisite thatthe refractive index n₃ of the cavity layer and the refractive index n₁of the substrate should both exceed the refractive index n₂ of thespacer layer. Also, since at least one mode must exist in the cavity toachieve resonance, the cavity layer must exceed a certain minimumthickness.

The cavity layer is preferably a thin-film of dielectric material.Suitable materials for the cavity layer include silicon nitride, hafniumdioxide, zirconium dioxide, titanium dioxide, aluminium oxide andtantalum oxide.

The cavity layer may be prepared by known techniques, eg vacuumevaporation, sputtering, chemical vapour deposition, plasma-enhanced orplasma-impulse chemical deposition, or in-diffusion.

The dielectric spacer layer must have a lower refractive index than boththe cavity layer and the substrate. The layer may, for example, comprisean evaporated or sputtered layer of magnesium fluoride. In this case aninfra-red light injection laser may be used as light source. The lightfrom such a source typically has a wavelength around 600-800 nm. Othersuitable materials include lithium fluoride and silicon dioxide. Apartfrom the evaporation and sputtering techniques mentioned above, thespacer layer may be deposited on the substrate by a sol-qel process, orbe formed by chemical reaction with the substrate.

The sol-gel process is particularly preferred where the spacer layer isof silicon dioxide.

The refractive index of the substrate (n₁) must be greater than that(n₂) of the spacer layer but the thickness of the substrate is generallynot critical.

By contrast, the thickness of the cavity layer must be so chosen thatresonance occurs within an appropriate range of coupling angles. Thespacer layer will typically have a thickness of the order of severalhundred nanometres, say from about 200 nm to 2000nm, more preferably 500to 1500 nm, eg 1000 nm. The cavity layer typically has a thickness of afew tens of nanometres, say 10 to 200 nm, more preferably 30 to 150 nm,eg 100 nm.

It is particularly preferred that the cavity layer has a thickness of 30to 150 nm and comprises a material selected from silicon nitride,hafnium dioxide, zirconium dioxide, titanium dioxide, tantalum oxide andaluminium oxide, and the spacer layer has a thickness of 500 to 1500 nmand comprises a material selected from magnesium fluoride, lithiumfluoride and silicon dioxide, the choice of materials being such thatthe refractive index of the spacer layer is less than that of the cavitylayer.

Preferred materials for the cavity layer and the spacer layer aresilicon nitride and silicon dioxide respectively.

At resonance, the incident light is coupled into the cavity layer byFTR, propagates a certain distance along the cavity layer, and couplesback out (also by FTR). The propagation distance depends on the variousdevice parameters but is typically of the order of 1 or 2 mm.

At resonance the reflected light will undergo a phase change, and it isthis which may be detected. Alternatively, as described in InternationalPatent Application No WO 92/03720 the cavity layer and/or spacer layermay absorb at resonance, resulting in a reduction in the intensity ofthe reflected light.

For use in the determination of biochemical species, the surface of thebiosensor device, ie the surface of the cavity layer in the case of anFTR sensor, will generally be sensitised by having biomolecules, egspecific binding partners for the analyte(s) under test, immobilisedupon it. The immobilised biochemicals may be covalently bound to thesensor surface by methods which are well known to those skilled in theart.

As described in International Patent Application No WO 92/21976, thebiosensor may be coated with a layer of a biocompatible porous matrixof, for example, dextran within which suitable binding molecules may beimmobilised.

The invention will now be described in greater detail, by way ofillustration only, with reference to the accompanying drawings, in which

FIG. 1 is a partial schematic diagram of an analytical apparatusaccording to the invention;

FIG. 2 is sectional side view of a sample cuvette and stirring mechanismforming part of the apparatus of FIG. 1;

FIG. 3 is an exploded perspective view of the sample cuvette of FIG. 2;

FIG. 4 is a sectional side view of the sample cuvette of FIG. 3;

FIG. 5 is a schematic end view of the sample cuvette of FIG. 3, showinghow the cuvette is clamped in position; and

FIG. 6 is a perspective view of a stirrer element forming part of themechanism of FIG. 2;

FIG. 7 is a view similar to FIG. 2 of an analytical apparatus accordingto the invention incorporating a second form of stirrer mechanism;

FIG. 8 is a side view of a stirrer element for use in a third embodimentof an analytical apparatus according to the invention;

FIG. 9 is a plan view of the stirrer element of FIG. 8;

FIG. 10 is an end view of the stirrer element of FIG. 8; and

FIG. 11 is a plan view of a blank from which the stirrer element of FIG.8 is formed.

Referring first to FIG. 1, an analytical apparatus for the qualitativeor quantitative determination of a biochemical analyte is based on theprinciple of frustrated total reflection.

The apparatus comprises a sample cuvette 1 which defines a samplechamber 2. The base of the chamber 2 is a glass block 3, the uppersurface of which is coated with several layers of material.

As shown in the enlarged portion of FIG. 1, the first layer applied tothe surface of the block 3 is a relatively low refractive index layer 31of silica with a thickness of approximately 500 nm. The second layer 32is relatively high refractive index hafnia, of thickness approximately100 nm. The hafnia layer is in turn coated with a layer of dextran 33within which antibodies or other biomolecules are immobilised in knownfashion.

The multilayer structure at the upper surface of the block 3 constitutesan FTR biosensor. The interface between the block 3 and the silica layer31 is irradiated with light from a laser light source 4, which can bemoved such that the angle of incidence of the light can be varied over acertain range. The hafnia layer 32 acts as a resonant cavity and at acertain angle of incidence of the light the evanescent field created byreflection at the interface between the block 3 and the silica layer 31is coupled into the hafnia layer 32. The angle at which resonance occursdepends on the refractive index in the vicinity of the hafnia layer 32and this is modified by interaction between the immobilised antibodiesand analyte molecules (represented in FIG. 1 by solid black dots) in asample which is introduced into the sample chamber 2.

Movement of the laser light source 4 over a range of angles is carriedout as described in our International Patent Application No WO 93/14391.A polariser 6 is interposed between the light source 4 and the block 3,and is arranged such that the light incident on the block 3 hasapproximately equal TE (transverse electric) and TM (transversemagnetic) components. An analyser 5 is disposed between the block 3 anda detector 7 such that reflected light reaches the detector 7 only whenresonance occurs. FIG. 1 shows a typical plot of measured lightintensity I versus angle of incidence θ. There are two resonant angles:one for TM-polarised light and the other for TE-polarised light.

The sample contained within the sample chamber 2 is stirred by a stirrerelement 8 mounted on the end of a stirrer shaft 9. The shaft extendsinto the chamber 2 and, in used, the element 8 reciprocates along anaxis perpendicular to the surface of the block 3, as shown by thedouble-headed arrow in FIG. 1.

The sample cuvette 1 and stirrer mechanism are shown in greater detailin FIG. 2, and the cuvette 1 also in FIGS. 3 and 4.

Referring next to FIG. 3, the sample cuvette 1 comprises an aluminiumbody produced by pressure die-casting. The body has a surface coating ofdeposited electroless nickel. The cuvette 1 is formed with laterallyextending wings 22,23 which, in use, serve to support the cuvette 1 andprovide for good thermal contact between the cuvette 1 and the adjacentsurfaces of the measuring apparatus.

The cuvette 1 includes a recess 24 which receives a twin sample well 25.The well 25 is used for the storage of analytical reagents such asbuffer solutions.

A pair of downwardly-depending limbs 26,27 receive a moulded siliconerubber gasket 28 and the glass block 3, the upper surface of which iscoated as described above. The block 3 is held in place by uv-curedadhesive 29 applied around its base.

The silicone gasket 28 defines a sample compartment 40 with a volume ofapproximately 250μl. Access to the compartment 40 is via a well 46formed in the top of the body 21. The well 46 comprises a centralportion 43 of circular cross-section and two diametrically opposedextension channels 44,45 (see FIG. 4).

In an alternative embodiment, the gasket 28 may be omitted and the block3 secured directly to the underside of the cuvette 1 between the limbs26,27. The sample compartment is then defined by the lower region of thewell 46.

As can be seen from FIG. 5, when the cuvette 1 is positioned on themeasuring apparatus, a clamping block 36 acts through twodownwardly-depending members 37,38 on the wings 22,23 to hold themagainst an aluminium block 41 which forms part of the apparatus.

In addition to clamping the cuvette 1 in position, the block 36 alsohouses and supports the stirrer mechanism.

Referring now to FIG. 6, the stirrer element 8 comprises a 120°frusto-conical plate 61, formed by deformation of a 3.5 mm diameterstainless steel disc with a 1.1 mm diameter central aperture. The plate61 is connected by a stirrup 62, formed from 0.25 mm thickness stainlesssteel sheet, to a lower drive shaft 63. The stirrup 62 is spot-welded toboth the plate 61 and the lower drive shaft 63.

Referring once again to FIG. 2, the lower drive shaft 63 has a diameter,at the point where it is connected to the stirrup 62, of 0.5 mm. At itsupper end, the lower drive shaft 63 is broadened to a diameter of 2 mm.The lower drive shaft 63 threadedly engages the lower end of an upperdrive shaft 64 which extends through a bore in the clamping block 36,and through a retaining plate 70 into an upper motor unit.

The upper drive shaft 64 is connected at its upper end to a coil formerassembly comprising a coil mounting plate 66 and an upwardly extending,hollow, cylindrical coil former 67. A disc spring 68 is captivatedbetween the coil mounting plate 66 and the upper drive shaft 64, and isheld at its periphery between the retaining plate 70 and a connectionguide 69.

To prevent twisting of the disc spring 68 and coil former assembly whenthe lower drive shaft 63 is threadedly engaged with or disengaged fromthe upper drive shaft 64, the upper portion of the upper drive shaft 64has a square cross-section, corresponding to the shape of the centralaperture in the retaining plate 70.

The retaining plate 70 and connection guide 69 fit closely within anupper motor case carriage 71, and retain a magnet assembly and O-ring77. The magnet assembly comprises a backing plate 72, to the undersideof which are fixed, by adhesive, a central button magnet 73 and an outerring magnet 74. A soft iron inner core 75 is fixed to the underside ofthe button magnet 73 and an annular soft iron outer core 76 is fixed byadhesive to the underside of the ring magnet 74.

The coil former 67, on which the coil is wound, is freely mounted aboutthe button magnet 73/soft iron inner core 75. The backing plate 72, thesoft iron outer core 76 and the connection guide 69 are provided withperipheral slots 79,80,81 through which flexible connections are led tothe coil.

In use, an alternating current of frequency 120 Hz is applied to thecoil. This causes the coil former assembly, and hence the stirrerelement 8, to reciprocate along the axis of the upper and lower driveshafts 63,64 through an amplitude of ±0.3 mm. The rest position of thestirrer element 8 is approximately 0.5 mm above the coated surface ofthe block 3, and thus the stirrer element 8 oscillates between extremepositions 0.2 mm and 0.8 mm above that surface.

A sample is injected into the sample compartment 40 by means of asyringe inserted into one of the extension channels 45. The otherextension channel 44 receives a suction needle 42 which is used toremove sample from the compartment 40 after a measurement is complete.

The cuvette 1 is positioned on the supporting aluminium block 41.

Sample is introduced into the compartment 40 either before or after thecuvette is so positioned. The block 3 is irradiated with light from thelight source 4, such that the light is reflected towards the detector 7.The angle of incidence of the light is varied and the angle at whichresonance occurs (detected as a peak in the light reaching the detector)is determined.

The stirring of the sample by the stirrer element 8 during measurementensures homogenisation of the sample and prevents any spurious effectsdue to transport phenomena within the sample. The good thermal contactbetween the wings 22,23 of the cuvette 1 and the supporting block 41ensures rapid thermal equilibration, as does the fact that the thermalcapacity of the cuvette 1 is very much greater than that of the sample.

FIG. 7 shows an arrangement which is generally very similar to that ofFIG. 2, save that the stirrer element 85 is rotated rather thanvibrated.

Finally, FIGS. 8 to 11 show a form of stirring element 90 which isintended for use in an apparatus comprising two adjacent samplechambers. The element 90 comprises a beam 91 which is fixed at one endby means of a flange 92. The beam 91 is, in use, fixed to the end of areciprocating stirrer shaft (shown by the broken line in FIG. 8) suchthat the free end of the beam 91 vibrates, as shown by the double-headedarrow in FIG. 8. The stirrer shaft is driven by an electromagneticmechanism similar to that shown in FIG. 2. The free end of the beam 91is formed with downwardly depending limbs 93,94, the ends of which areforked and splayed. The limbs 93,94 extend, in use, into two adjacentsample compartments in a cuvette generally similar to that describedabove.

The stirrer element 91 is formed from the blank shown in FIG. 11, bytwisting of the beam 91 in the region of the line A--A and splaying ofthe forked ends of the limbs 93,94 along the line B--B.

I claim:
 1. An analytical apparatus comprising a biosensor device, asample chamber having a base incorporating a sensitized surface of thebiosensor device, a stirrer extending into the sample chamber, and meansfor causing the stirrer to vibrate within the sample chamber with areciprocating motion.
 2. Apparatus as claimed in claim 1, wherein thestirrer comprises an elongate stirrer shaft which terminates at a pointwithin the sample chamber which, in use, lies within a sample fluidcontained in the sample chamber.
 3. Apparatus as claimed in claim 1,wherein the stirrer comprises an elongate stirrer shaft connected to afurther component which intrudes into the sample chamber.
 4. Apparatusas claimed in claim 1, wherein the means for causing the stirrer to movewithin the sample chamber comprises an electric or electromagneticmotor.
 5. Apparatus as claimed in claim 1, wherein a portion of thestirrer which, in use, is immersed in the sample fluid is provided withan element to facilitate mixing of the sample fluid, said element beingin the form of a hollow truncated cone, the top and bottom faces ofwhich are open.
 6. Apparatus as claimed in claim 1, in which the stirrercomprises an elongate stirrer shaft, the upper end of which is rigidlyconnected to a former on which is wound a wire coil to which analternating current may be applied.
 7. Apparatus as claimed in claim 6,wherein the shaft and the former constitute an assembly which is securedto a flexible membrane.
 8. Apparatus as claimed in claim 1, wherein thefrequency of reciprocation is 100-150 Hz.
 9. Apparatus as claimed inclaim 1, wherein the amplitude of reciprocation is less than ±0.5 mm.10. Apparatus as claimed in claim 1, wherein the sample chamber formspart of a disposable cuvette.
 11. Apparatus as claimed in claim 10,wherein the cuvette is of a material with high thermal conductivity. 12.Apparatus as claimed in claim 10, wherein the cuvette is of a materialwith high thermal conductivity which is a metal.
 13. Apparatus asclaimed in claim 11, wherein the material with high thermal conductivitycomprises aluminum.
 14. Apparatus as claimed in claim 10, wherein thecuvette is provided with wings or flanges by which the cuvette may behold in intimate thermal contact with a support.
 15. Apparatus asclaimed in claim 1, wherein the biosensor device is a frustrated totalreflection sensor comprisinga) a cavity layer of transparent dielectricmaterial of refractive index n₃, b) a dielectric substrate of refractiveindex n₁, and c) interposed between the cavity layer and the substrate,a dielectric spacer layer of refractive index n₂ wherein n₂ is less thann₁ and n₃.